The goal of this proposal is to determine the molecular mechanisms by which flaviviruses, notably the yellow fever vaccine strain YFV-17D and the pathogenic Asibi strain, as well as dengue virus (DENV), stimulate innate immunity to program adaptive immune responses. This broad goal will be pursued in 3 aims: Specific Aim 1: To determine the mechanisms by which ISR programs DCs to stimulate adaptive Immunity. Our recent use of systems biological approaches to understand immune responses to vaccination in humans has revealed an early transcriptional signature, consisting of mammalian general control nonderepressible-2 [GCN2], that is a strong correlate of the later CD8+ T cell response. GCN2 is a sensor of amino acid starvation, and mediates the integrated stress response (ISR) and translational control of mRNA. We will thus study the impact of the ISR and translational control in the innate control of adaptive immunity. Specific Aim 2: To evaluate the Innate and adaptive immune responses stimulated by YFV-17D, versus Aslbl. YFV-17D was derived from the Asibi strain, but very little is understood about the mechanisms of innate sensing and regulation of adaptive immunity by Asibi. Here we will use molecular clones of the two viruses to determine the signaling networks by which these two viruses program DCs and monocytes, and the impact of this on T and B cell immunity. Specific Aim 3: To determine the development and functions of human monocyte subsets In response to DENV. Our recent work demonstrates that during acute dengue infection, there is a substantial expansion in the CD14+CD16+ monocyte population. This subset seems to be efficient at inducing plasmablast differentiation from resting B cells. Here we will explore the immune stimulatory capacities and developmental programs of these cells. Sub-Aim 3a: To determine the Immune stimulatory capacity of monocytes stimulated by DENV; Sub-Aim 3b: Probing the gene regulatory networks driving differentiation of CD14+CD16+ monocytes The successful completion of these aims will offer novel insights into the mechanisms by which flaviviruses stimulate innate immunity, and guide the design of therapeutic and vaccination strategies.